The long-term goal of this project is to define the general principles and molecular mechanisms by which aquaporin water channels fold, integrate and assemble into the endoplasmic reticulum (ER) membrane. Aquaporins comprise a highly conserved protein family that plays a major role in normal and pathological water homeostasis in the kidney, lung, brain and other tissues. The molecular basis of aquaporin function is achieved by a precise arrangement of six transmembrane segments and two short helices in a two-fold inverted symmetry surrounding a monomeric water-selective pore. Recent studies have demonstrated that formation of this remarkable structure is orchestrated via precise and ordered interactions between the nascent polypeptide and the ribosome-translocon complex (RTC) ER. However, the underlying mechanisms by which the RTC facilitates aquaporin folding and is in turn regulated by aquaporin structure is only beginning to be understood. New approaches are therefore needed to examine nascent membrane proteins in their native folding environment. The studies outlined in this proposal will address two fundamental aspects of this process. First, they will directly define how the RTC facilitates aquaporin folding and membrane integration of transmembrane segments. Second they will determine how structural properties of aquaporins act in a reciprocal fashion to control RTC structure and function. The Specific Aims will: 1. Characterize structural and functional properties of the nascent polypeptide that control membrane integration and progression through the translocon. 2. Define the mechanism by which the ribosome translocon complex controls accessibility of the nascent polypeptide to different cellular compartments. 3. Define the timing and molecular environment of cotranslational AQP folding. Proposed experiments will use translationally incorporated probes to directly access the molecular environment of the nascent polypeptide within the RTC. Photocrosslinking to truncated functional translocation intermediates will define how structural features within the nascent AQP polypeptide control sequential stages of membrane integration during TM segment entry, progression, and exit from the Sec61 a translocon pore. Collisional quenching of incorporated fluorophores will determine the precise stage of synthesis at which lumenal and cytosolic peptide loops gain access to their appropriate cellular compartments. Finally, Forester Resonance Energy Transfer will be used to determine the stage of synthesis and location within the RTC at which a-helix formation takes place and early tertiary structure is formed. Together this combined biochemical and biophysical approach will define how the RTC facilitates early aquaporin folding, how 2x and 3x structure formation impacts interactions between the nascent polypeptide and RTC, and how these interactions regulate RTC structure to direct protein topology and membrane insertion. Results of these studies will provide a major advance in our understanding of normal and pathological mechanisms of polytopic protein folding.